Mass Spectroscopy

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Mass Spectroscopy
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A simple definition of a Mass Spectrometer

• A Mass Spectrometer is an analytical instrument
  that can separate charged molecules according to
  their masstocharge ratio.
• The mass spectrometer can answer the questions
  what is in the sample (qualitative structural
  information) and how much is present
  (quantitative determination) for a very wide
  range of samples at high sensitivity

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Components of a Mass Spectrometer
Vacuum System
Atmosphere
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How are mass spectra produced ?

• Ions are produced in the source and are
  transferred into the mass analyser
• They are separated according to their mass/charge
  ratio in the mass analyser (e.g. Quadrupole, Ion
  Trap)
• Ions of the various m/z values exit the analyser
  and are counted by the detector

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What is a Mass Spectrum ?

• A mass spectrum is the relative abundance of ions
  of different m/z produced in an ion source
• -a chemical fingerprint
• It contains
• Molecular weight information (generally)
• Structural Information (mostly)
• Quantitative information

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What does a mass spectrum look like ?

• Electrospray mass spectrum of salbutamol

240
100
Base peak at m/z 240

241
0
60
80
100
120
140
160
180
200
220
240
260
280
300
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What information do you need from the analysis ?

• Low or High Mass range
• Average or Monoisotopic mass (empirical)
• Accurate Mass
• Quantitation - precision, accuracy, selectivity
• Identification
• Structural Information
• Isotope Ratios

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Mass Analyzers

• Types of Mass Spectrometer
• Magnetic Sector
• Quadrupole
• Ion Trap
• Time-of-flight
• Hybrid- Sector/trap, Quad/TOF etc
• Mass Spectrometers separate ions according to
  their mass-to-charge (m/z) ratios

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A Triple Stage Quadrupole Mass Analyzer
Finnigan TSQ
Q2 is Non-Linear Collision Cell
Q0
Q1
Q2
Q3
ESI Probe
Square Rod Ion Transmission to Analytical Quads
Electron Multiplier, Detection System
Hyperbolic, high precision quadrupoles
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How does the quadrupole work ?
The quadrupole consists of four parallel rods.
The opposing rods have the same polarity whilst
adjacent rods have opposite polarity.
Each rod is applied with a DC and an RF
voltage.Ions are scanned by varying the DC/Rf
quadrupole voltages. Only ions with the selected
mass to charge ratio will have the correct
oscillatory pathway in the Rf field.
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Quadrupole Mass Analyzer

• The ion is transmitted along the quadrupole
  in a stable trajectory Rf field. The ion
  does not have a stable trajectory and is ejected
  from the quadrupole.

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Ion Trap

• Consists of ring electrode and two end caps
• Principle very similar to quadrupole
• Ions stored by RF DC fields
• Scanning field can eject ions of specific m/z
• Advantages
• - MS/MS/MS..
• - High sensitivity full scan MS/MS

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What is the advantage of using high resolution
mass spectrometry?
The advantage of high resolution measurement is
to eliminate chemical background of the same
nominal mass but different accurate mass and,
therefore to increase the signal to noise ratio
and the sensitivity respectively. Better
selectivity is obtained by applying high
resolution in case of isobaric compounds, i.e.
two compounds of same nominal mass but different
accurate mass. With low resolution only a
combined Spectral result is obtained under
Product ion conditions . With high resolution
separate detection and therefore separate
isolation and MS/MS spectra are
obtained. Example R 1000 R
5000 Compound mass1 372.351 Da
OVERLAPPING SEPARATION Compound mass2
372.421 Da
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Types of ionization techniques

• Volatile samples
• Electron ionization
• Chemical ionization
• GC (and LC) inlets

• Non-volatile samples
• Fast Atom Bombardment
• Thermospray
• Matrix Assisted Laser Desorption ionization
• Electrospray ionization
• Atmospheric Pressure Chemical ionization
• LC (and GC) inlets

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Comparison of ionization Techniques
200,000
15,000
1,000
Molec. Weight
Non Polar
Highly Polar
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Electron ionization

• Widely used technique when coupled to GC
• Suitable for volatile organic compounds
• eg hydrocarbons, oils, flavours, fragrances
• Not really coupled to LC today
• Also called electron impact

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Electron ionization

• Produces M. radical cation giving molecular
  weight
• Produces abundant fragment ions
• Library searchable spectra
• Energetic process. A heated filament emits
  electrons which are accelerated by a potential
  difference of usually 70eV into the sample
  chamber. ionization of the sample occurs by
  removal of an electron from the molecule thus
  generating a positively charged ion with one
  unpaired electron.

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Electron ionization
Filament
Extraction lenses
Sample Inlet
Collector
Source magnets
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Electron ionization

• M e- M. 2e-
• M. A
  B
• A
• M.
• B

Fragmentation
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Chemical ionization

• Development from EI
• Same compound classes as EI
• Gives molecular weight
• Softer ionization technique
• Produces MH ions or M - H- ions
• Used to produce more abundant molecular ions when
  the molecule under investigation fragments using
  EI

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Chemical ionization

• Similar ionization technique to EI except that a
  reagent gas is introduced into the chamber in
  excess of the sample
• Positive CI uses methane, isobutane or ammonia as
  reagent gases
• Negative CI uses methane reagent gas in electron
  capture mode
• Ionised reagent gas protonate the sample
  molecules leaving a neutral reagent gas species
• Not reproducible from lab to lab, hence no CI
  libraries.

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Chemical ionization
Extraction lenses
Filament
Sample Inlet
Collector
Source magnets
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Chemical ionization
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Fast Atom Bombardment

• Used for large compounds with low volatility (eg
  peptides, proteins, carbohydrates)
• Solid or liquid sample is mixed with a
  non-volatile matrix (eg glycerol, crown ethers,
  nitrobenzyl alcohol)
• Immobilised matrix is bombarded with a fast beam
  of Argon or Xenon atoms. Charged sample ions are
  ejected from the matrix and extracted into the
  mass analysers
• Gives MH or MNa ions
• Choosing correct matrix is difficult

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FAB Source
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Matrix Assisted Laser Desorption ionization

• Similar process to FAB
• Sample is dissolved in matrix which absorbs light
  from a short pulse of laser of a specific
  wavelength. The sample becomes ionised and
  extracted towards the mass analysers
• Coupled to Time of Flight MS
• Not coupled to LC
• High mass range achievable
• Calibrants may be external or included in sample
• Reproducibility issues

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Thermospray

• First widely used LC/MS interface
• Flow rates 0.5 - 1.5 ml/min
• Good for polar compounds
• LC eluent containing sample and ammonium acetate
  is pumped through a heated vaporiser. The jet of
  vapour contains small charged droplets which
  evaporate under the heat and vacuum expelling
  charged ions from the surface
• Produces MH or M - H- ions
• Not commercially available today

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Thermospray Process
Thermospray nozzle
Solvent evaporation due to heat and reduced
pressure
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Atmospheric Pressure ionization

• Most important and widely used LC / MS technique
• API two types
• Electrospray
• Atmospheric Pressure Chemical ionization
• gt 99 new LC/MS use API source
• ionization takes place outside vacuum region

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Atmospheric Pressure ionization

• API coupled to LC or CE or Nanospray
• Handle wide range of flow rates
• Produce Intense MH ions
• Very little fragmentation
• Need MS/MS for structural information
• Applicable to wide range of compounds
• Sample must be in solution

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Electrospray

• Electrospray also known as
• Ionspray
• Nanospray
• Sonic Spray
• Pure Electrospray
• ESI, ES, IS

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Electrospray

• Softest ionization technique
• Best for polar non-volatile compounds (proteins,
  peptides, nucleic acids, Pharmaceuticals, natural
  products)
• Coupled to LC at a flow range of 2-1000 ul/min,
  nanospray (10 nL/min 2 uL/min)
• Ions are ejected from charged vapour droplets to
  gas phase producing MH or M - H- ions
• Can produce multiply charged ions allowing
  determination of high molecular weight proteins
• Not very tolerant of non-volatile salts

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ESI Probe
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Electrospray Process
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APCI

• Atmospheric Pressure Chemical ionization, also
  known as
• APCI
• Heated nebulizer
• APcI

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APCI

• Used for wide range polarity of compounds
• HPLC eluent (up to 2ml/min flow rate) is
  vaporised at up to 600 oC
• The Corona discharge needle ionises solvent
  molecules. A combination of collisions and charge
  transfer reactions between the solvent and the
  analyte results in the transfer of a proton to
  form either MH or M-H- ions
• Compounds can thermally degrade
• Multiply charged ions rare
• More tolerant to salts

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APCI Probe
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APCI Process
LC eluent evaporated from heated vaporiser
Corona discharge needle ionises solvent to
generate a chemical ionization reagent gas plasma
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Solvent suitability

• HPLC buffers
• Reversed phase most often used
• MeOH, ACN, H2O,
• TFA, formic acid, acetic acid, Ammonium formate,
  ammonium acetate
• Normal phase can be used
• Non-volatile buffers
• OSA, aQa self cleaning source, off-axis probe

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Problems

• How would you analyse this compound ?
• Naphthalene
• What sample introduction technique could you use
  ? Which ionization technique ?
• A EI, GC/MS

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Problems

• How would you analyse this compound ?
• Phenacetin
• What sample introduction technique could you use
  ? Which ionization technique ?
• A API (either APCI or ESI), LC/MS

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Problems

• How would you analyse myoglobin ?
• Myoglobin is a protein with a molecular weight of
  16,951.
• If the Mass Spectrometer has a mass range of up
  to 4,000, how can you analyse high molecular
  weight proteins ?

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Multiply charged myoglobin ions from ESI
(M2-1.008) /M1-M2 Z1 (Z1 M1)-(Z1.008) Mwt
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Deconvoluted myoglobin spectrum
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Biomolecular Applicationsof LC/MS
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Overview

• Biomolecule LC/MS basics
• mass spectrometry (MS) instrumentation
• triple quads, ion traps, MS, MS/MS, MSn
• electrospray ionization (ESI)
• mass range, multiple charging
• Peptide sequencing
• database searching for protein i.d.
• De novo sequencing (a more advanced topic)
• Methods and Applications
• Case studies
• Protein i.d. from gel-separated proteins
• Applications in biopharmaceuticals
• Automation strategies
• Sample cleanup
• On-line digestion
• On-line interaction screening
• Data-handling strategies

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Information Obtainable via ESI/LC/MSn

• Molecular Weight
• confirmation of sequence
• screening for post-translational or other
  covalent modifications
• heterogeneity/impurity profiling
• Structural Information (via MS/MS or MSn)
• sequence confirmation
• protein identification from database searching of
  fragment ions
• disulfide bond mapping
• determination of sites of modification or
  mutation
• Binding/Interaction
• on-line affinity selection or affinity
  chromatography

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Challenges/limitations of LC/MS for Biomolecular
Analysis

• ESI/MS of intact proteins can be difficult if
  protein is too heterogeneous
• most protein i.d. requires enzyme digestion first
• detergents interfere with LC/MS analysis and must
  be removed which is non-trivial
• low tolerance of ESI to salt and 100 aqueous
  solns makes analysis of proteins in their native
  state difficult
• Information and data overload!

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LC/Electrospray Ionization/MS
Electrospray ionization (ESI) separation of ions
from bulk solution so that they may be analyzed
in the gas phase (mass spectrometry)
charged aerosol formation (1 atm)
desolvation, ion evaporation (rough vacuum)
Mass Analysis (vacuum)
Ion focusing (med vacuum)
20 uL/min
5kV
HPLC desalting, concentration, separation of
proteins and peptides
u-HPLC and autosampler
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Schematic Diagram of a Triple Quadrupole Mass
Spectrometer
A tandem-in-space mass spectrometer different
stages of mass analysis performed in discrete
regions (quads 1 or 3)
Q2 is Non-Linear Collision Cell
Q0
Q1
Q2
Q3
ESI Probe
Electron Multiplier, Detection System
Hyperbolic, high precision quadrupoles
Square Rod Ion Transmission to Analytical Quads
strengths great general purpose system MS,
MS/MS, neutral loss and parent scans useful for
mixture screening, best instrument for intact
protein analysis, no low mass cut-off in MS/MS
mode like the ion trap, data-dependent
scanning limitations less sensitive than ion
trap in the full scan MS/MS mode
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Proteins require enzymatic cleavage before
sequencing can occur by MS
Enzyme Digest
Separation (HPLC)
Database Searching
Fragmentation MS/MS
Sequence ions
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Fragmentation in MS/MS produces sequence-specific
fragment ions

• peptides fragment along the amide backbone to
  produce sequence specific fragment ions
• different patterns of sequence ions are produced
  from the different masses of the side chain
  groups
• ions containing the N-terminus are typically of
  type a or b
• ions containing the C-terminus are known as y
  ions
• tryptic peptides predominantly produce ions of
  type y due to the presence of Arg or Lys at the
  C-terminus

a
b
O
O
R1
R2
COH
C
CH
CH
N
H2N
H
y

• Use database search routines to identify or
  profile known proteins
• De novo sequencing requires interpretation of
  spectra either manually or by computer algorithm